1: PROJECT SUMMARY Breathing is a complex behavior that is fundamental for life in all mammals. Disturbances in the function and coordination of breathing are common in many disorders of the central nervous system. Thus, the study of specific neural populations that underlie this behavior is not only of great basic science interest, but holds high clinical significance. Lesion experiments and in-vitro studies using transverse brainstem slices have defined the minimal circuitry that is necessary and sufficient for the inspiratory phase of breathing, a small ?kernel? of neurons in the ventrolateral medulla termed the preBtzinger Complex (preBtC). Excitatory neurons derived from cells expressing the transcription factor Dbx1 are thought to form the rhythmogenic ?core? of the preBtC. However, a ?refractory period? for Dbx1 stimulation following each breath limits the ability these neurons to drive a high frequency rhythm in-vitro. The role of this specific population of neurons in controlling the wide range of breathing frequencies common in-vivo is unknown. In this project we will use novel in-vivo and in-vitro approaches conducted in parallel to investigate the role of Dbx1 neurons in the generation of inspiration when embedded in the wider medullary network. Based on our preliminary data, we hypothesize that the inspiratory neural network functions as a distributed column, and is not limited to a defined ?core? region. Inhibition of excitatory Dbx1 neurons effectively drives the inspiratory rhythm through post-inhibitory rebound. And, the refractory period for Dbx1 can be reduced in the distributed inspiratory network to allow faster breathing frequencies by modulating mechanisms of short-term synaptic depression. These hypotheses will be tested using powerful optogenetic, electrophysiological, pharmacological and imaging techniques in anesthetized and freely behaving mice (Aim1) and in a novel horizontal brainstem slice preparation that preserves the wider medullary network bilaterally (Aim2). We expect that integration of these preparations will provide a unique perspective to examine issues that remain unresolved in the field of respiratory rhythm generation.